Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for formalin-fixed and paraffin-embedded (FFPE) tissue - Part 3: Isolated DNA (ISO 20166-3:2018)

This document gives guidelines on the handling, documentation, storage and processing of formalin-fixed and paraffin-embedded (FFPE) tissue specimens intended for DNA examination during the pre-examination phase before a molecular assay is performed.
This document is applicable to molecular in vitro diagnostic examinations including laboratory developed tests performed by medical laboratories and molecular pathology laboratories. It is also intended to be used by laboratory customers, in vitro diagnostics developers and manufacturers, biobanks, institutions and commercial organizations performing biomedical research, and regulatory authorities.
NOTE International, national or regional regulations or requirements can also apply to specific topics covered in this document.

Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für präanalytische Prozesse für formalinfixierte und paraffineingebettete (FFPE)-Gewebeproben - Teil 3: Isolierte DNS (ISO 20166-3:2018)

Dieses Dokument dient als Leitfaden zur Handhabung, Dokumentation, Lagerung und Verarbeitung von aus FFPE Gewebe bestehendem und für die DNA Untersuchung vorgesehenem Untersuchungsmaterial während der präanalytischen Phase vor Beginn der molekularen Analyse.
Dieses Dokument gilt für molekulare in vitro diagnostische Untersuchungen, wozu auch im Labor entwickelte Prüfungen zählen, die von medizinischen Laboratorien und Laboratorien der molekularen Pathologie durch¬geführt werden. Sie soll auch von Laborkunden, Entwicklern und Herstellern von In vitro Diagnostika sowie Biobanken, Einrichtungen und kommerziellen Organisationen, die in der biomedizinischen Forschung tätig sind, und Aufsichtsbehörden eingesetzt werden.
ANMERKUNG   Zu bestimmten Bereichen, die in diesem Dokument behandelt werden, können lokale, nationale oder regionale Bestimmungen oder Anforderungen gelten.

Analyses de diagnostic moléculaire in vitro - Spécifications relatives aux processus préanalytiques pour les tissus fixés au formol et inclus en paraffine (FFPE) - Partie 3: ADN extrait (ISO 20166-3:2018)

Le présent document fournit des lignes directrices concernant la manipulation, la documentation, le stockage et le traitement de prélèvements de tissus fixés au formol et inclus en paraffine (FFPE) destinés à l'analyse de l'ADN durant la phase préanalytique précédant la réalisation d'une analyse moléculaire.
Le présent document s'applique aux analyses de diagnostic moléculaire in vitro, y compris les analyses développées en laboratoire, réalisées par des laboratoires de biologie médicale et des laboratoires de pathologie moléculaire. Il est également destiné à être utilisé par des clients de laboratoires, des développeurs et fabricants de l'industrie du diagnostic in vitro, ainsi que par des biobanques, des institutions et des organismes commerciaux spécialisés en recherche biomédicale, de même que des autorités de réglementation.
NOTE Des réglementations ou exigences internationales, nationales ou régionales peuvent également s'appliquer à des sujets spécifiques traités dans le présent document.

Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne procese za tkiva, ki so fiksirana v formalinu ter položena v parafin - 3. del: Izolirani DNK (ISO 20166-3:2018)

Ta mednarodni standard vsebuje priporočila za obravnavo, dokumentiranje, shranjevanje in obdelavo vzorcev tkiv, ki so fiksirana v formalinu ter položena v parafin (FFPE), namenjenih za analizo DNK med predpreiskovalno fazo, preden se izvede molekularni preskus. Ta mednarodni standard se uporablja za molekularne diagnostične preiskave in vitro, vključno z laboratorijsko razvitimi preskusi, ki jih izvajajo v medicinskih laboratorijih in laboratorijih za molekularno patologijo. Uporabljali naj bi ga tudi uporabniki laboratorijev, razvijalci in proizvajalci diagnostike in vitro, nanaša pa se tudi na institucije in komercialne organizacije, ki izvajajo biomedicinske raziskave, biobanke ter regulativne organe. Celovitost DNK v tkivih se lahko spremeni pred fiksacijo v formalinu, obdelavo in shranjevanjem ter med temi postopki. Kemijske spremembe DNK med fiksacijo tkiva lahko privedejo do fragmentacije in sprememb zaporedja, sprememb v stanju metilacije ali celo strukturnih sprememb, kar lahko vodi do npr. lažnih sprememb števila kopij pri primerjalni genomski hibridizaciji z uporabo mikromrež (aCGH). Te spremembe molekul DNK lahko vplivajo na veljavnost in zanesljivost rezultatov preskusov preiskav. Zato je nujno treba sprejeti posebne ukrepe, da se zmanjšajo opisane spremembe za nadaljnje preiskave DNK. OPOMBA:   Za določene teme, ki so zajete v tem mednarodnem standardu, lahko veljajo tudi mednarodni, nacionalni ali regionalni predpisi ali zahteve.

General Information

Status
Published
Publication Date
22-Jan-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
23-Jan-2019
Completion Date
23-Jan-2019

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SLOVENSKI STANDARD
SIST EN ISO 20166-3:2019
01-maj-2019
1DGRPHãþD
SIST-TS CEN/TS 16827-3:2015
0ROHNXODUQHGLDJQRVWLþQHSUHLVNDYHLQYLWUR6SHFLILNDFLMH]DSUHGSUHLVNRYDOQH

SURFHVH]DWNLYDNLVRILNVLUDQDYIRUPDOLQXWHUSRORåHQDYSDUDILQGHO,]ROLUDQL

'1. ,62

Molecular in vitro diagnostic examinations - Specifications for pre-examination processes

for formalin-fixed and paraffin-embedded (FFPE) tissue - Part 3: Isolated DNA (ISO

20166-3:2018)
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für formalinfixierte und paraffineingebettete (FFPE)-
Gewebeproben - Teil 3: Isolierte DNS (ISO 20166-3:2018)

Analyses de diagnostic moléculaire in vitro - Spécifications relatives aux processus

préanalytiques pour les tissus fixés au formol et inclus en paraffine (FFPE) - Partie 3:

ADN extrait (ISO 20166-3:2018)
Ta slovenski standard je istoveten z: EN ISO 20166-3:2019
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
SIST EN ISO 20166-3:2019 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST EN ISO 20166-3:2019
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SIST EN ISO 20166-3:2019
EN ISO 20166-3
EUROPEAN STANDARD
NORME EUROPÉENNE
January 2019
EUROPÄISCHE NORM
ICS 11.100.10 Supersedes CEN/TS 16827-3:2015
English Version
Molecular in vitro diagnostic examinations - Specifications
for pre-examination processes for formalin-fixed and
paraffin-embedded (FFPE) tissue - Part 3: Isolated DNA
(ISO 20166-3:2018)

Analyses de diagnostic moléculaire in vitro - Molekularanalytische in-vitro-diagnostische Verfahren

Spécifications relatives aux processus préanalytiques - Spezifikationen für präanalytische Prozesse für

pour les tissus fixés au formol et inclus en paraffine formalinfixierte und paraffineingebettete (FFPE)-

(FFPE) - Partie 3: ADN extrait (ISO 20166-3:2018) Gewebeproben - Teil 3: Isolierte DNS (ISO 20166-

3:2018)
This European Standard was approved by CEN on 21 December 2018.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20166-3:2019 E

worldwide for CEN national Members.
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SIST EN ISO 20166-3:2019
EN ISO 20166-3:2019 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 20166-3:2019
EN ISO 20166-3:2019 (E)
European foreword

This document (EN ISO 20166-3:2019) has been prepared by Technical Committee ISO/TC 212 "Clinical

laboratory testing and in vitro diagnostic test systems" in collaboration with Technical Committee

CEN/TC 140 “In vitro diagnostic medical devices” the secretariat of which is held by DIN.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by July 2019, and conflicting national standards shall be

withdrawn at the latest by January 2022.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

This document supersedes CEN/TS 16827-3:2015.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,

France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,

Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and the United Kingdom.
Endorsement notice

The text of ISO 20166-3:2018 has been approved by CEN as EN ISO 20166-3:2019 without any

modification.
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SIST EN ISO 20166-3:2019
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SIST EN ISO 20166-3:2019
INTERNATIONAL ISO
STANDARD 20166-3
First edition
2018-12
Molecular in vitro diagnostic
examinations — Specifications for pre-
examination processes for formalin-
fixed and paraffin-embedded (FFPE)
tissue —
Part 3:
Isolated DNA
Analyses de diagnostic moléculaire in vitro — Spécifications relatives
aux processus préanalytiques pour les tissus fixés au formol et inclus
en paraffine (FFPE) —
Partie 3: ADN extrait
Reference number
ISO 20166-3:2018(E)
ISO 2018
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SIST EN ISO 20166-3:2019
ISO 20166-3:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
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SIST EN ISO 20166-3:2019
ISO 20166-3:2018(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2  Normative references ...................................................................................................................................................................................... 1

3  Terms and definitions ..................................................................................................................................................................................... 1

4  General considerations .................................................................................................................................................................................. 4

5  Outside the laboratory ................................................................................................................................................................................... 5

5.1 Specimen collection ............................................................................................................................................................................ 5

5.1.1 General...................................................................................................................................................................................... 5

5.1.2 Information about the specimen donor/patient .................................................................................. 5

5.1.3 Information about the specimen ....................................................................................................................... 5

5.1.4 Specimen processing .................................................................................................................................................... 6

5.2 Transport requirements ................................................................................................................................................................. 6

6  Inside the laboratory ....................................................................................................................................................................................... 7

6.1 Information about the reception of the specimen .................................................................................................... 7

6.2 Formalin fixation of the specimen or sample(s) ........................................................................................................ 7

6.3 Evaluation of the pathology of the specimen and selection of the sample(s) .................................. 8

6.4 Post-fixation of frozen samples ................................................................................................................................................ 9

6.5 Decalcification ......................................................................................................................................................................................... 9

6.6 Processing and paraffin embedding ..................................................................................................................................... 9

6.7 Storage requirements .....................................................................................................................................................................10

6.8 Isolation of DNA ..................................................................................................................................................................................10

6.8.1 General...................................................................................................................................................................................10

6.8.2 General information for DNA isolation procedures ........................................................................10

6.8.3 Using commercial kits ..............................................................................................................................................11

6.8.4 Using the laboratories’ own protocols .......................................................................................................11

6.9 Quantity and quality assessment of isolated DNA ................................................................................................12

6.10 Storage of isolated DNA ................................................................................................................................................................12

Annex A (informative) Impact of the storage temperature on DNA Integrity in FFPE blocks

of tissue .......................................................................................................................................................................................................................14

Bibliography .............................................................................................................................................................................................................................16

© ISO 2018 – All rights reserved iii
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SIST EN ISO 20166-3:2019
ISO 20166-3:2018(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso

.org/iso/foreword .html.

This document was prepared by Technical ISO/TC 212, Clinical laboratory testing and in vitro diagnostic

test systems.
A list of all parts in the ISO 20166 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2018 – All rights reserved
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SIST EN ISO 20166-3:2019
ISO 20166-3:2018(E)
Introduction

Molecular in vitro diagnostics, including molecular pathology, has enabled significant progress in

medicine. Further progress is expected with new technologies analysing nucleic acids, proteins, and

metabolites in human tissues and body fluids. However, the profiles and/or integrity of these molecules

can change drastically during specimen collection, transport, storage and processing, thus making

the outcome from diagnostics or research unreliable or even impossible because the subsequent

examination assay will not determine the situation in the patient but an artificial molecular pattern

generated during the pre-examination process. Studies have been undertaken to determine the

influencing factors for the DNA examination from formalin-fixed and paraffin-embedded (FFPE) tissue.

These studies demonstrated that a standardization of the entire process from specimen collection to the

DNA examination is needed. This document draws upon such work to codify and standardize the steps

for FFPE tissue with regard to DNA examination in what is referred to as the pre-examination phase.

DNA integrity in tissues can change before, during and after formalin fixation, processing and storage.

Chemical modifications introduced into DNA during tissue fixation might lead to fragmentation and

sequence alterations, changes in the methylation status or even structural changes which can lead to,

for instance, spurious copy number changes in array-CGH profiles. These modifications of the DNA

molecules can impact the validity and reliability of the examination test results. Therefore, it is essential

to take special measures to minimize the described DNA changes and modifications for subsequent

examination.
In this document, the following verbal forms are used:
— "shall" indicates a requirement;
— "should" indicates a recommendation;
— "may" indicates a permission;
— "can" indicates a possibility or a capability.
© ISO 2018 – All rights reserved v
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SIST EN ISO 20166-3:2019
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SIST EN ISO 20166-3:2019
INTERNATIONAL STANDARD  ISO 20166-3:2018(E)
Molecular in vitro diagnostic examinations —
Specifications for pre-examination processes for formalin-
fixed and paraffin-embedded (FFPE) tissue —
Part 3:
Isolated DNA
1 Scope

This document gives guidelines on the handling, documentation, storage and processing of formalin-

fixed and paraffin-embedded (FFPE) tissue specimens intended for DNA examination during the pre-

examination phase before a molecular assay is performed.

This document is applicable to molecular in vitro diagnostic examinations including laboratory

developed tests performed by medical laboratories and molecular pathology laboratories. It is also

intended to be used by laboratory customers, in vitro diagnostics developers and manufacturers,

biobanks, institutions and commercial organizations performing biomedical research, and regulatory

authorities.

NOTE International, national or regional regulations or requirements can also apply to specific topics

covered in this document.
2  Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 15189:2012, Medical laboratories — Requirements for quality and competence
ISO 15190, Medical laboratories — Requirements for safety
3  Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 15189 and the following apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
aliquot

portion of a larger amount of homogeneous material, assumed to be taken with negligible sampling error

Note 1 to entry: The term is usually applied to fluids. Tissues are heterogeneous and therefore cannot be

aliquoted.
Note 2 to entry: The definition is derived from References [25], [26], and [27].
© ISO 2018 – All rights reserved 1
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SIST EN ISO 20166-3:2019
ISO 20166-3:2018(E)
3.2
ambient temperature
unregulated temperature of the surrounding air
3.3
analyte
component represented in the name of a measurable quantity
[SOURCE: ISO 17511:2003, 3.2, modified — EXAMPLE has been removed.]
3.4
analytical test performance

accuracy, precision, specificity and sensitivity of a test to measure the analyte (3.3) of interest

Note 1 to entry: Other test performance characteristics such as robustness, repeatability can apply as well.

3.5
cold ischemia

condition after removal of the tissue from the body until stabilization or fixation

3.6
diagnosis

identification of a health or disease state from its signs and/or symptoms, where the diagnostic process

can involve examinations (3.8) and tests for classification of an individual's condition into separate and

distinct categories or subclasses that allow medical decisions about treatment and prognosis to be made

3.7
DNA
deoxyribonucleic acid

polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded

(ssDNA) form
[SOURCE: ISO 22174:2005, 3.1.2]
3.8
examination
analytical test

set of operations having the object of determining the value or characteristics of a property

Note 1 to entry: Processes that start with the isolated analyte and include all kinds of parameter testing or

chemical manipulation for quantitative or qualitative examination.

[SOURCE: ISO 15189:2012, 3.7, modified — Notes to entry 1 to 3 have been removed, Note 1 to entry has

been added and “analytical test” has been added as a preferred term.]
3.9
formalin

saturated aqueous formaldehyde solution which at 100 % contains 37 % formaldehyde by mass

(corresponding to 40 % by volume)
3.10
formalin fixation

treatment of a sample (3.18) with standard buffered formalin solution (3.20) for stabilization

3.11
grossing
gross examination

inspection of pathology specimens with the bare eye to obtain diagnostic information, while being

processed for further microscopic examination
2 © ISO 2018 – All rights reserved
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SIST EN ISO 20166-3:2019
ISO 20166-3:2018(E)
3.12
interfering substances

endogenous substances of a specimen (3.15)/sample (3.18) or exogenous substances (e.g. stabilization

solution) that can alter an examination result
3.13
paraffin embedding

process in which a tissue sample is placed in paraffin to generate a hard surrounding matrix so that

thin microscopic sections can be cut
3.14
pre-examination process
pre-analytical phase
pre-analytical workflow

process that starts, in chronological order, from the clinician’s request and includes the examination

request, preparation and identification of the patient, collection of the primary sample(s) (3.15),

transportation to and within the medical or pathology laboratory, isolation of analytes (3.3), and ends

when the analytical examination begins

Note 1 to entry: The pre-examination phase includes preparative processes that influence the outcome of the

intended examination.

[SOURCE: ISO 15189:2012, 3.15, modified — “pre-analytical workflow” has been added as a preferred

term, Note 1 to entry has been added and the definition has been extended.]
3.15
primary sample
specimen

discrete portion of a body fluid, breath, hair or tissue taken for examination (3.8), study or analysis of

one or more quantities or properties assumed to apply for the whole

[SOURCE: ISO 15189:2012, 3.16, modified — Notes to entry 1 to 3 have been removed.]

3.16
proficiency test

evaluation of participant performance against pre-established criteria by means of inter-laboratory

comparisons

[SOURCE: ISO 17043:2010, 3.7, modified — Notes to entry 1 and 2 have been removed.]

3.17
room temperature
for the purposes of this document, temperature in the range of 18 °C to 25 °C
Note 1 to entry: Local or national regulations can have different definitions.
3.18
sample
one or more parts taken from a primary sample (3.15)
[SOURCE: ISO 15189:2012, 3.24, modified — EXAMPLE has been removed.]
3.19
stability

ability of a sample material, when stored under specified conditions, to maintain a stated property

value within specified limits for a specified period of time
Note 1 to entry: The analyte for the purpose of this document is isolated DNA.

[SOURCE: ISO Guide 30:2015, 2.1.15, modified — “reference material” has been replaced by “sample

material" and Note 1 to entry has been changed.]
© ISO 2018 – All rights reserved 3
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SIST EN ISO 20166-3:2019
ISO 20166-3:2018(E)
3.20
standard buffered formalin solution
neutral buffered formalin
NBF

10 % formalin (3.9) solution in water with a mass fraction of 3,7 % (corresponding to a volume fraction

of 4 %) formaldehyde, buffered to pH 6,8 to pH 7,2

Note 1 to entry: Standard buffered formalin solutions often contain small amounts of methanol to inhibit

oxidation and polymerisation of formaldehyde.
3.21
storage

prolonged interruption of the pre-analytical workflow (3.14) of a sample (3.18) or analyte (3.3)

respectively, or of their derivatives, such as stained sections or tissue blocks, under appropriate

conditions in order to preserve their properties

Note 1 to entry: Long-term storage typically occurs in laboratory archives or in biobanks.

3.22
tissue processor

automated instrument where tissue fixation, dehydration, clearing and paraffin infiltration occurs

3.23
validation

confirmation, throughout the provision of objective evidence, that the requirements for a specific

intended use or application have been fulfilled
Note 1 to entry: “Validated” is used to designate the corresponding status.

[SOURCE: ISO 9000:2015, 3.8.13, modified — Notes to entry 1 and 3 have been removed.]

3.24
warm ischemia

condition before the tissue is removed from the body, but where it is deprived of its normal blood supply

3.25
workflow
series of activities necessary to complete a task
3.26
homogeneous
uniform in structure and composition
4  General considerations

For general statements on medical laboratory quality management systems and in particular on

specimen collection and handling (including avoidance of cross contaminations) see ISO 15189:2012,

4.2, 5.4.4, 5.4.6 or ISO/IEC 17020:2012, clause 8 and 7.2. The requirements for laboratory equipment,

reagents, and consumables in accordance with ISO 15189:2012, 5.3 shall be followed; ISO 15189:2012,

5.5.1.2 and 5.5.1.3, and ISO/IEC 17020:2012, 6.2 can also apply.

All steps of a diagnostic workflow can influence the final analytical test result. Thus, the entire

workflow including biomolecule stability and sample storage conditions shall be verified and validated.

Workflow steps which cannot always be controlled (e.g. warm ischemia) shall be documented. A risk

assessment of non-controllable workflow steps including their potential impact on the analytical test

performance shall be performed and mitigation measures shall be established to enable the required

analytical test performance.

In contrast to RNA or proteins, DNA in tissue is relatively stable during warm and cold ischemia.

Changes of DNA sequence or copy numbers [e.g. comparative genomic hybridization (CGH) profiles] due

4 © ISO 2018 – All rights reserved
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SIST EN ISO 20166-3:2019
ISO 20166-3:2018(E)
[7]

to longer warm and cold ischemia durations are unknown . However, DNA methylation patterns may

[6]

change in response to ischemia . The duration until the specimen is placed into standard buffered

formalin solution should be kept as short as possible in order to avoid enzymatic degradation of DNA.

The duration before fixation shall be documented and the temperature before fixation should be

[5]
documented .

During the fixation, processing and storage, the DNA integrity can change depending on the kind

of fixative, fixation time and temperature, storage or archiving of the fixed paraffin-embedded

tissue as well as the method used for DNA isolation and purification. When using a fixative based on

formaldehyde, temperature and fixation duration have a significant impact on DNA integrity. The longer

the fixation duration and the higher the temperature, the more chemical modifications and crosslinks

[9][10][11][12][13][14]

are introduced, which can lead to degradation or sequence alterations . These effects

can limit the size of amplifiable target DNA and/or influence the target sequence of primers used for

amplification.

Safety instructions on specimen transport and handling shall be considered and followed in accordance

with ISO 15189:2012, 5.2.3 and 5.4.5 and ISO 15190.

During the whole pre-examination process precautions shall be taken to avoid cross contamination

between different specimens/samples, e.g. by using single-use material whenever feasible or

appropriate cleaning procedures between processing of different specimens/samples.

If a commercial product is not used in accordance with the manufacturers' instructions, responsibility

for its use and performance lies with the user.
5  Outside the laboratory
5.1  Specimen collection
5.1.1  General

For the collection of the specimen, the requirements (e.g. disease condition, specimen size) for intended

molecular examination (see also Clause 6) should be considered.
See also ISO 15189:2012, 5.4.4.
5.1.2  Information about the specimen donor/patient

The documentation shall include the ID of the specimen donor/patient, which can be in the form of a code.

The documentation should include, but is not limited to:

a) the relevant health status of the specimen donor/patient [e.g. healthy, disease type, concomitant

disease, demographics (e.g. age and gender)];

b) the information about routine medical treatment and special treatment prior to tissue collection

(e.g. anaesthetics, medications, surgical or diagnostic procedures);
c) the appropriate consent from the specimen donor/patient.
5.1.3  Information about the specimen
The documentation shall include, but is not limited to:

a) the start of ischemia within the body (warm ischemia) by documentation of the ischemia-relevant

vessel ligation/clamping time point (usually arteria
...

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